PROJECT SUMMARY The colonic epithelium forms the first cellular barrier between the complex microbial milieu of the colonic lumen and the body. How the epithelium senses and responds to this environment is critical to protecting the body and controlling inflammation. Hydrogen peroxide (H2O2) is an important signaling molecule in cells and is involved in epithelial repair and host defense. Studies have shown that patients with early-onset inflammatory bowel disease and colon cancer have impaired regulation of H2O2. Extracellular H2O2 is produced by cell- membrane oxidases (e.g. NADPH oxidase 1 (NOX1)), secreted oxidase enzymes, and commensal bacteria within the gut. The mechanism by which H2O2 in the intestinal environment signals to the epithelium of the intestine is poorly understood and represents an important gap in knowledge. Aquaporin 3 (AQP3), one of the aquaporin family of plasma membrane channels, is known to conduct H2O2 and is highly expressed in the colonic epithelium. My previous studies show that H2O2 transport through AQP3 is important for epithelial repair after injury and for the inflammatory response to a pathogenic microbe. However, the mechanisms by which AQP3 modulates these innate immune functions is not well understood. The overall aim of this proposal is therefore to understand how AQP3-regulated H2O2 signaling might shape epithelial responses to injury and microbes ? critical processes in the gut that are important for inflammatory and infectious intestinal disease. Aim 1 of my proposal investigates how H2O2 transport through AQP3 affects signal transduction in the colonic epithelium and its role in wound repair. Specifically, I will examine: how the source and location of the H2O2 signal impacts AQP3-dependent cell signaling; if AQP3 is co-regulated with NOX1; and whether AQP3 amplifies wound repair by H2O2-producing commensal microbes. Aim 2 investigates how AQP3-mediated H2O2 transport impacts epithelial inflammatory responses to pathogenic and commensal bacteria. Specifically, I will be looking at whether a pathogen (c.rodentium) induces NOX1 generation of H2O2 and induces inflammatory signaling via AQP3, and whether associated Toll-like receptor activation or a H2O2-producing commensal bacterium can alter AQP3-mediated inflammatory signaling. To carry out these aims, wild-type (AQP3+/+/ NOX1+/+), AQP3-/- and NOX1-/- mice and primary colonic enteroids will be used. Fluorescence imaging will be used to measure H2O2 and an endoscopic injury model will be used in vivo to assess wound repair. Activation of cellular signaling will be analyzed using protein expression and biotinylation studies. The information arising from this proposal may provide new therapeutic targets for treatment of intestinal inflammatory disorders and lead to a better understanding of the role of aquaporins at the mucosal surface of the intestine. The results may also apply more broadly to how H2O2 is regulated at other microbe-colonized surfaces of the body, such as the urinary tract, skin, and airways, and therefore may have an impact beyond gastrointestinal disease.